INTRODUCTION The accurate replication of duplex DNA requires the coordinated activity of many types of proteins. We are using a multienzyme system of phage T4 proteins to determine the nature of the proteins, enzymatic reactions, and protein-protein interactions that catalyze and control this intricate and essential process. T4 DNA polymerase, attached to a sliding clamp protein, catalyzes DNA synthesis on both leading and lagging strands. The gene 41 helicase moves 5' to 3' on the lagging strand template, opening the duplex ahead of the leading strand polymerase, and interacting with the primase to allow it to make the RNA primers that initiate lagging strand synthesis. Although the helicase can load on nicked and forked DNA by itself, its loading is greatly accelerated by the 59 helicase loading protein, particularly on DNA that is covered by the gene 32 ssDNA binding protein. The RNA primers and adjacent DNA are ultimately removed by a T4 encoded 5' to 3' nuclease (T4 RNase H), and following gap repair, the adjacent fragments are joined by T4 DNA ligase. The same proteins carry out origin-dependent replication early in infection, and recombination-dependent replication at later times. HELICASE AND LOADING PROTEIN- During the past year we have continued our studies of the mechanism by which the 59 helicase-loading protein binds to 32 protein, and loads the helicase. We find that 59, a tight fork binding protein, promotes the binding of 32 protein on single-stranded fork arms that are too short for tight cooperative binding of 32 alone. The helicase loader remains on the DNA after 32 protein is added. On forks long enough for 32 protein to bind alone, it shows a clear preference for binding to the fork arm corresponding to the lagging strand template, suggesting that there may be polarity in the assembly of cooperatively bound 32 protein on ssDNA. The crystal structure of 59 helicase loading protein, previously solved by our collaborators Timothy Mueser and Craig Hyde, revealed a novel, almost entirely -helical protein, whose N-terminal domain has strong structural similarity to HMG family proteins. We proposed a speculative model for 59 protein on a replication fork DNA, based on the assumption that the "HMG-like" N-domain binds the duplex ahead of the fork and holds the beginning of the fork arms in an open configuration to provide docking areas for 41 helicase and 32 protein. We are now engaged in an extensive mutagenesis and deletion analysis to identify essential 59 residues and test this model. REPLICATION FROM T4 ORIGINS- Two bacteriophage T4 origins of replication, (ori(uvsY) and ori(34)), have been shown by Ken Kreuzer and coworkers to consist of a T4 middle-mode promoter juxtaposed to a DNA unwinding element (DUE). In collaboration with Kreuzer (Duke University) we established a system of T4 replication proteins that catalyze complete unidirectional replication of a plasmid containing the T4 ori(uvsY) origin, with a preformed R loop at the position of the R loop identified at this origin in vivo. We showed that this replication is dependent on the 41 helicase and strongly stimulated by 59 protein, and that the helicase loading protein helps to coordinate leading and lagging strand synthesis by blocking replication on the ori(uvsY) R loop plasmid until the helicase is loaded. Our current efforts are directed at determining where and when the first lagging strand begins in vitro, and what experimental parameters influence how far the leading strand elongates in the absence of topoisomerase. STRUCTURE AND FUNCTION OF T4 RNaseH- T4 RNaseH is a 5' to 3' exonuclease that is a member of the RAD2 family of replication and repair nucleases. We previously showed that this nuclease removes the RNA primers and 10-50 nucleotides of adjacent DNA from each discontinuous lagging strand fragment on the DNA replication fork in vitro, and that the extent of this degradation is controlled by the differential effect of 32 protein on the nuclease and lagging strand polymerase. The C-terminal region of the nuclease is required for this interaction with 32 protein. We find that T4 RNaseH is also stimulated by the polymerase clamp, and that hydrophobic residues at the N-terminus of the nuclease are necessary for this stimulation. In the crystal structure of RNaseH, the N- and C- termini are close together behind the catalytic site of the nuclease, placing both the clamp and 32 protein in the proper orientation to circle or bind to the ssDNA behind the nuclease. We are collaborating with Timothy Mueser (University of Toledo) in efforts to determine the crystal structures of the nuclease with DNA and with 32 protein. We have recently shown that the crystal structure of metal free T4 RNaseH is more ordered than our previous structure of the nuclease with Mg. The bridge above the active site, present in structures of related proteins, but disordered in native T4 RNase H, is clearly seen in the metal free structure. STRUCTURE OF REPLICATION FORKS- We are collaborating with Jack Griffith (University of North Carolina) to characterize the path of DNA and the structure of proteins on T4 replication forks by electron microscopy. Helicase, unwinding the duplex ahead of the fork, will lengthen the region of ssDNA behind the last fragment on the lagging strand. There will be a second region of ssDNA ahead of this fragment, if the fragment is still being elongated by polymerase. In the trombone model of replication, proposed by Bruce Alberts, the elongating new fragment and the ssDNA behind it are folded into a loop, so that the leading and lagging strand polymerases can remain together. The proposed loop would grow in size as the fragment is elongated, disassemble when the polymerase reaches the beginning of the previous fragment, and form again when polymerase begins elongating the next primer. In our electron micrographs of products of the T4 replication system, one or two regions of ssDNA are visible, as expected, on deproteinized replication forks. Micrographs of forks with the replication proteins show the predicted DNA loop and large protein complexes at the fork, but unexpectedly the loops are completely double-stranded. There is no extended ssDNA on these replicating molecules. Our studies suggest that all of the ssDNA on the lagging strand is in a compact, protein-covered form within the large complexes. This architecture is likely to be an important feature of replication forks, because ssDNA has been shown by others to be compacted at forks with phage T7 proteins. We have hypothesized that this compact structure could be a factor in controlling when Okazaki fragments start, and shielding the most recent primer from nuclease degradation. We are now using biotin labeled-proteins to ask what proteins are present in the large complexes we have visualized at the fork, and where within the complexes they are located. We have used streptavidin beads attached to a short rigid dsDNA as visible pointers to show polymerase, helicase, 59 helicase loading protein, and 32 protein within the complexes on molecules with extensive replication. It is likely that the structure of the compacted ssDNA within these complexes is determined by 59 helicase loading protein as well as 32 protein, because the structure appears to be less compact in forks from reactions without 59 protein.